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Methods Mol Biol. 2018;1832:339-356. doi: 10.1007/978-1-4939-8663-7_19.

Probing the Function of Oncohistones Using Mutant Transgenes and Knock-In Mutations.

Author information

1
Department of Pediatrics, Institute for Cancer Genetics, Columbia University, New York, NY, USA.
2
Department of Genetics and Development, Institute for Cancer Genetics, Columbia University, New York, NY, USA.
3
Department of Neurosurgery, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China.
4
Department of Pediatrics, Institute for Cancer Genetics, Columbia University, New York, NY, USA. zz2401@cumc.columbia.edu.
5
Department of Genetics and Development, Institute for Cancer Genetics, Columbia University, New York, NY, USA. zz2401@cumc.columbia.edu.

Abstract

Recently, frequent somatic mutations at histone genes have been detected in high grade pediatric brain tumor, chondroblastoma, and giant cell tumor of bone. These mutant histones are also termed oncohistones. Since oncohistone proteins co-exist with wild type histone proteins in cells, it is critically important to understand how they promote tumorigenesis. Here, we describe two methods to analyze the impact of these oncohistones on histone modification and epigenome, including the expression of oncohistone from a transgene and the utilization of CRISPR/Cas9 system to knock-in specific oncohistone mutations. The methods described are useful for the initial characterization of oncohistones. Other methods such as ChIP-seq and RNA-seq, which analyze the effect of oncohistone mutations genome wide, are not detailed in this protocol.

KEYWORDS:

Acid extraction; CRISPR/Cas9; ChIP-seq and RNA-seq; Histone marks; Immunofluorescence; Oncohistone; Western Blot

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